Universal carrier for targeting molecules to Gb3 receptor expressing cells

ABSTRACT

The present invention concerns an universal polypeptidic carrier for targeting directly or indirectly a molecule to Gb3 receptor expressing cells and having the following formula STxB-Z(n)-Cys, wherein: STxB is the Shiga Toxin B subunit or a functional equivalent thereof, Z is an amino-acid devoided of sulfydryl group, n being 0, 1 or a polypeptide, Cys is the amino-acid Cysteine, and the use thereof for MHC class I and MHC class II presentation of antigens.

[0001] The invention relates to a universal polypeptidic carrier fortargeting molecules to a Gb3 receptor for the B-subunit of Shiga-Toxinexpressing cells and its use for intracellular transport and processingof said molecules.

[0002] Shiga Toxin is a bacterial toxin of the AB₅ subunit family thatis secreted by Shigella dysenteriae. The A-subunit is the toxic moietyand inhibits the protein synthesis in higher eucaryotic target cellsafter transfering into the cytoplasm of said cells. The B-subunit is anhomopentamer protein (5B-fragments) and is responsible for toxin bindingto and internalization into target cells by interacting with theglycolipid Gb3 found on the plasma membranes of these cells. TheB-fragment is non toxic, but conserves the intracellular transportcharacteristics of the holotoxin which, in many Gb3 expressing cells, istransported in a retrograde fashion from the plasma membranes tocytosol, via endosomes.

[0003] The glycolipid Gb3 receptor has also been reported to beexpressed preferentially in some ectodermic derived tumors (plasma) andsome Burkitt's lymphoma. It is also known as CD 77. In the present text,the term Gb3 should be considered as an equivalent to CD77.

[0004] The authors have already shown that a CD8 human tumor Antigenfused to the B subunit of Shiga toxin could efficiently be presented inan HLA class 1-restricted manner to specific CTL (1). This result wasindependently confirmed by another study that demonstrated that Shigaholotoxin, carrying a defined peptide epitope from influenza virus,could deliver the antigen into the MHC class I intracellular pathway(3).

[0005] The authors have also shown that fusion proteins between the Gb3receptor-binding non toxic B-fragment of bacterial Shiga toxin derivedfrom Shigella dysnteriae and an antigen, or an epitope from a modeltumor antigen, can elicit specific cytotoxic T lymphocytes response(CTL), whereas each moiety of said fusion protein does not leadindividually to CTL induction (1, 2, and WO 99/03881).

[0006] The difficulty of this technology is that, for each application,i.e., for each antigen or fragment thereof, there is a need for aspecific construction of a fusion protein, that necessitates a specificconstruction of a recombinant vector bearing the sequences encoding thisfusion protein to be expressed in a host cell.

[0007] The aim of the present invention is to overcome theabove-mentioned drawbacks and to provide a universal hook, or auniversal carrier for targeting a molecule to a Gb3 receptor expressingcell to enable this molecule to be internalized, processed and/orexpressed in said cell expressing Gb3 receptor.

[0008] In the present invention, a Shiga toxin B-subunit (STxB)derivative, or mutant, termed STxB-Cys has been designed. In thisprotein, a Cysteine is added at the C-terminus of mature STxB. Theprotein, when purified from bacteria, carries the internal disulfidebond, as wild type STxB, while the sulfhydryl group at the C-terminalCys is free. Due to their nucleophilicity, free sulfhydryl groups areexcellent acceptors for directed coupling approaches (4).

[0009] Thus, the present invention relates to a universal polypeptidiccarrier for targeting directly or indirectly a molecule of interest toGb3 receptor expressing cells having the following formula:STxB-Z(n)-Cys, wherein:

[0010] STxB is the Shiga Toxin B subunit or a functional equivalentthereof,

[0011] Z is an amino-acid devoided of sulfydryl group, n being 0, 1 oran amino-acid sequence,

[0012] Cys being the amino-acid Cysteine.

[0013] The STxB moiety of the universal carrier has the sequencedescribed in (8) or a functional equivalent thereof. A functionalequivalent means a polypeptidic sequence having the capacity to bindspecifically to the Gb3 receptor and/or to trigger an internalization ofan antigen and its presentation in an MHC class-I restricted pathway, orboth MHC class I and class II on the same antigen presenting cell.

[0014] In the light of the heterogeneity of expression of tumorantigens, the allele-specific loss of MHC class I expression at thesurface of tumor cells, and the necessity to have concomitantpresentation of antigens by both MHC class I and class II on the sameantigen presenting cell, it is advantageous to couple full size antigenproteins to the B-subunit for targeting to dendritic cells.

[0015] In a preferred embodiment, n is 0 and the universal carrier hasthe following sequence (SEQ ID No 1): COOH -MKKTLLIAASLSFFSASALATPDCVTGKVE YTKYNDDDTFTVKVGDKELFTNRWNLQSLLLSAQITGMTVTIKTNACHNGGGFSEVIFRC - NH2

[0016] As a matter of fact, if the Z linker is too long, i.e., when n isequal or greater than 2, some internal disulfide bridges might occur,and prevent either the binding of STxB to the Gb3 receptor andespecially prevent the binding to the molecule of interest.

[0017] According to the invention, the molecule of interest is selectedin the group constituted of proteins, peptides, oligopeptides,glycoproteins, glycopeptides, nucleic acids, polynucleotides, or acombination thereof.

[0018] In another aspect of the invention, the molecule of interest isan antigen to be targeted to antigen presentating cells. Such cells areselected in a group comprising T lymphocytes, dendritic cells,macrophages Langerhans cells and the like.

[0019] In another aspect of the invention, the molecule of interest aredrugs such as haptenes, psoralenes, or any compounds provided that theyhave a chemical group linkable with the —SH group of the Cysteine moietyof STxB-Cys.

[0020] The drug might be linked either directly or after activation withcoumpounds such as bromoacetate, or any other method known by a skilledperson, provided that the result of the reaction is a chemical entityhaving the following formula: STxB-Cys-M, M being all the abovementioned molecules of interest.

[0021] The coupling approaches for covalent binding of a peptidic or apolypeptidic moiety to STxB-Z(n)-Cys can be any method or processesdescribed or carried out by a skilled person.

[0022] A first method that can be embodied is the use of SPDPhetero-bi-functional cross-linker described par Carlsson et al (5).However, SPDP is capable of being cleavable by serum thiolases that is acause of decreasing the yield of the reaction.

[0023] A second method for covalent coupling of STxB-Z(n)-Cys peptideswith another peptide of interest is to produce bromoacetyl or maleimidefunctions on the latter as described by P. Schelte et al (4). Briefly,the peptide of interest is chemically activated with bromoacetateanhydride or by a maleimide group respectively. In appropriate reactionconditions (pH, temperature, incubation times), these groups areeliminated by cis-elimination, yielding respectively to —S—S, —S—CH₂—,to —S—CO— or to —S—NH— covalents linkages.

[0024] As an example, the polypeptide or the peptide to be coupled tothe —SH moiety the C-terminal Cysteine of the universal carrier, has itsN-terminus activated with bromoacetic anhydride following the reactionscheme:

[0025] The Bromoacetyl function has high chemoselectivity for peptidethiol groups and the activated peptide can be reacted with STxB-Cys asfollows:

[0026] STxB-Cys-SH+Br—CH₂—CO—NH-peptide

STxB-Cys-S—CH₂—CO—NH-peptide+HBr

[0027] The resulting thioether-linkage is stable to hydrolysis.

[0028] Another method for coupling a molecule to the universal carrierof the invention is to use MBS (m-Maleimidobenzoyl-N-hydroxysuccinimideester) as shown in FIG. 6 and explained in example 5. This couplingallows the transport and processing of large molecules such antigenicproteins or glycoproteins through MHC class I and/or MHC class IIpathways.

[0029] Thus, another aspect of the invention is the product resultingfrom a covalent binding of STxB-Z(n)-Cys with a molecule of interest bya —S—S—, —S—CO—, or S—CH₂— or —S—NH— linkage.

[0030] In one embodiment, the molecule of interest to be targeted to anantigen presentating cells is constituted by or comprises a polypeptidicstructure, such an antigens or epitopes thereof, glycopeptides orglycoproteins, lipopeptides or lipoproteins.

[0031] In a preferred embodiment, the product resulting from thecoupling of STxB-Z(n)-Cys with an antigen or a fragment thereof, where(n) is 0, 1, or 2, and preferably 0, is able to be presented in an MHCclass I and MHC class II restricted pathway.

[0032] In another embodiment, the molecule of interest is a polypeptidecapable of binding with polynucleotide structures such as DNA or RNAmolecules. Such molecules might be vectors or plasmids comprising asequence of interest to be expressed in a target cell. In the presentinvention, a target cell is a eucaryotic cell bearing on its membranethe Gb3 receptor.

[0033] Thus, the universal carrier of the present invention is also acarrier for introducing a nucleotide sequence in a target cell eitherfor gene therapy or for obtaining recombinant cells expressingheterologous proteins.

[0034] In another embodiment, the universal carrier according to thepresent invention can be operably linked directly through a covalentbinding or indirectly through a linker to a cytotoxic drug to betargeted to tumor cells expressing Gb3 receptor.

[0035] The term “indirect binding” means that the universal carrier iscovalently linked through the sulfhydryl moiety of the C-terminalCysteine to a linker, said linker being operably linked to a drug or apro-drug to be internalized into Gb3 receptor bearing cells.

[0036] This linkage might be a covalent binding or a non-covalentbinding, provided that the affinity between the linker and the drug (orthe pro-drug) is higher than 10⁻⁹ mole/l.

[0037] Another aspect of the invention is an isolated polynucleotideselected from the group of:

[0038] (a) a polynucleotide comprising the nucleotide sequence STxBencoding the Shiga Toxin B subunit or a functional equivalent thereofbearing at its 3′end the codon TGT, or the codon TGC encoding Cysteine;

[0039] b) a polynucleotide comprising a nucleotide sequence having atleast 80% sequence identity to a nucleotide sequence encoding the ShigaToxin B subunit or a functional equivalent thereof bearing at its 3′endthe codon TGT or TGC; and

[0040] c) a nucleotide sequence complementary to the sequence in a) orb).

[0041] In a preferred embodiment, the polynucleotide has the followingSEQ ID No 2: 5′-atgaaaaaaacattattaatagctgcatcgctttcatttttttcagcaagtgcgctggcgacgcctgattgtgtaactggaaaggtggagtatacaaaatataatgatgacgatacctttacagttaaagtgggtgataaagaattatttaccaacagatggaatcttcagtctcttcttcttcagtgcgcaattacggggatgactgtaaccattaaaactaatgcctgtcataatggagggggattcagcgaagttatttttcgttgt-3′

[0042] The present invention relates also to a recombinant vector or toa plasmid comprising a polynucleotide sequence as described above, andcapable of expressing the universal carrier STxB-Z(n)-Cys, where (n) is0, 1 or 2, STxB and Z have the same significance as above, in anappropriate host cell.

[0043] As an example, a convenient vector is the plasmid pSu108described in (7).

[0044] Another object of the present invention is to provide a methodfor obtaining a plasmid expressing STxB-Z(n)-Cys comprising:

[0045] a) providing a plasmid comprising a STxB sequence;

[0046] b) applying two PCR amplification steps using two couples ofprimers, AA′ and BB′,

[0047] A and B being complementary to each other and comprising the Cyscodon,

[0048] A′ and B′ being outside the STxB sequence;

[0049] c) isolating the amplified fragments;

[0050] d) hybridizing the amplified fragments;

[0051] e) applying a PCR amplification on the hybridized fragments;

[0052] f) insertion of the amplified fragment into a plasmid.

[0053] In a preferred embodiment, the plasmid pSU108 (7) containing STxBfragment was modified to introduce the Cysteine codon TGT at the 3′ endof the B-fragment cDNA. The primers for step b) are respectively for AA′and BB′:

[0054] primer A: 5′-AGCGAAGTTATTTTTCGTTGTTGACTCAGAATAGCTC-3′ (SEQ ID 3),and

[0055] primer B: 5′-GAGCTATTCTGAGTCAACACGAAAAATAACTTC-3′ (SEQ ID no 4).

[0056] primer A′: primer ShigaAtpE: 5′-CACTACTACGTTTTAMC-3′ (SEQ ID no5), and

[0057] primer B′: primer Shiga-fd: 5′-CGGCGCAACTATCGG-3′ (SEQ ID no 6).

[0058] The PCR of step e) yields a fragment that is cloned into the SphIand SalI restriction sites of pSU108. Sequences derived by PCR areverified by dideoxy-sequencing.

[0059] The skilled person can easily design the choice of primers,plasmids for producing a vector bearing the polynucleotide sequenceexpressing STxB-Z(n)-Cys in an appropriate host cell, provided that thissuccession of steps allows the interpretation of the Cys codon into theamplified fragment.

[0060] The invention also provides a recombinant cell line obtained bytransformation with the recombinant vector containing the polypeptidesequence encoding the universal carrier as described above. In apreferred embodiment, said recombinant cell line is a procaryotic cell,preferentially E. coli.

[0061] In a still preferred embodiment, the plasmid is pSU108 having SEQID No. 2 integrated between the SphI and SalI restriction sites, and thecorresponding cell line has been deposited at CNCM on Dec. 19, 2000 withthe registration number 1-2604.

[0062] The present invention also provides a process for producing auniversal carrier as described above comprising:

[0063] a) culturing a recombinant cell line as described above,

[0064] b) obtaining a periplasmic extract of said cells, and

[0065] c) purifying said polypeptide.

[0066] Preferentially, the cell line is E. coli and in c) thepurification is made by anion exchange column chromatography followed bya gel filtration column chromatography.

[0067] Such a process is particularly advantageous for large scaleproduction of the universal carrier, as far as it can then be operablylinked by covalent coupling with a molecule of interest, and used withina large scope of application.

[0068] The present invention also provides a method for delivering asequence of interest into the MHC class I pathway using a productobtained by covalent binding of the Cys moiety of the universal carrierwith said sequence of interest; this method is advantageous to elicit aCTL respons to a given antigen or epitope thereof as far as the productis specific to the cell involved in the MHC class I pathway.

[0069] As a matter of fact, the inventors have shown that animmunodominant peptide, derived from the ovalbumin protein, and coupledchemically to STxB-Cys, could be presented by antigen presenting cellsto specific hybridoma cells, demonstrating that STxB could deliverexogenous immunogenic peptide in the MHC class I pathway. To exclude abias due to the presence of free peptides contaminating the material,experiments using fixed dendritic cells clearly demonstrated that theinternalization of the fusion protein was required for this process. Theinventors also have shown that the Shiga toxin receptor, Gb₃, was alsoinvolved in the ability of STxB-Cys to target exogenous peptide in theendogenous MHC class I pathway.

[0070] The invention also pertains to a method for delivering anexpression vector containing a sequence of interest into a Gb3 receptorexpressing cells characterized in that said expression vector is boundto a lysine rich peptide covalently linked to the Cys moiety of theuniversal carrier.

[0071] As an example, the lysine rich peptide is a 16-mer poly-lysinewhich is able to bind any polynucleotidic sequence, either of DNA or RNAnature. Such a peptide carrying a 16-mer of lysines will be activated atits N-terminus by bromoacetate anhydride and coupled to STxB-Cys.Expression plasmids will be bound to this coupling product, andvectorization of DNA into target cells is assayed using convenientreporter systems, such as the green fluorescent protein or luciferase.

[0072] The capacity to target expression plasmids with the help of STxBto the nucleus of antigen presenting cells is expected to furtherimprove the power of this vector, since i) DNA can even more easily beadopted to new experimental or clinical needs, and ii) due to itspotentiation effect, expression of antigenic peptides or proteins fromDNA would further increase the sensitivity of STxB-dependent antigenpresentation.

[0073] The invention also provides a method for delivering a drug or apro-drug into a cell, particularly into a cancer cell bearing Gb3 (orCD77) receptor.

[0074] The glycolipid Gb₃ receptor has been reported to bepreferentially expressed in some neuroectodermic derived tumors (glioma)and some Burkift's lymphoma. Since one limitation of the use ofchemotherapy in cancer is secondary side effects of the drugs because oftheir toxicity on normal cells, the drugs are preferentially vectorizedin tumor cells by using STxB-Cys. The drugs are activated to becomereactive with the sulfhydryl group of STxB-Cys. To achieve this, amaleimide group can be introduced on a drug, for example psoralenescompounds.

[0075] The present invention also pertains to:

[0076] a pharmaceutical composition for enhancing the immunogenicity ofa peptide or a protein or a glycoprotein or a lipopeptide, containingthe universal carrier covalently linked by its Cys moiety to saidpeptide or protein or glycoprotein or lipopeptide;

[0077] a pharmaceutical composition for treating tumor cells bearing theGb3 receptor (CD77), containing the universal carrier according to theinvention covalently linked by its Cys moiety to a drug or a pro-drugtoxic for said tumor cells.

[0078] Without limiting the scope of the universal carrier of theinvention and its widespread use in different applications, thehereinafter examples and figures illustrate the advantages of thepresent invention.

[0079] LEGEND OF THE FIGURES

[0080]FIG. 1 represents the protein profile of the final SephaDex 75column yielding purified STxB-Cys. Fractions 20-25 contain mostlymonomeric STxB-Cys (the positions of monomeric and dimeric STxB-Cys areindicated to the right). Molecular weight markers are indicated to theleft.

[0081]FIG. 2a represents th coupling of Type2 of Pep2 [as defined inexample 2] to STxB-Cys, followed by an in vitro antigen presentationassay on D1 dendritic cells, as described in (2). Two differentpreparations of STxB-Cys coupled to the SL8 peptide, an immunodominantepitope of ovalbumin were used (termed 4A and 9A). Upon fixation,antigen presentation is abolished showing that no extracellularprocessing occured.

[0082]FIG. 2b represents a control experiment of FIG. 2a, in which it isshown that fixed D1 can still present free SL8 peptide.

[0083]FIG. 3 represents another experiment on fixed and non fixed Dlcells using a coupling reaction of Type1 on STxB-SH and Pep1 [as definedin example 2].

[0084]FIG. 4 represents the B-subunit dependent presentation ofantigenic peptides derived from a coupling of Pep2 to B-Glyc-Cys-KDEL.The use of Fab fragments from an antibody that neutralizes STXB bindingto Gb3 also abolishes antigen presentation.

[0085]FIG. 5 shows that the Gb3 synthesis inhibitor PPMP inhibitsB-subunit dependent antigen presentation (5 a) and that SL8 presentationis not decreased in PPMP treated cells.

[0086]FIG. 6 represents reaction scheme for full size Ova coupling toSTxB-Cys using the heterobifunctional cross linker MBS. Top: firstreaction linking MBS to primary amines of Ova. Middle: second reactionbetween activated Ova and STxB-Cys. Bottom: Structure of MBS.

[0087]FIG. 7 shows western analysis of Ova coupling to STxB-Cys. Theupper part of the figure represents an immunoblot using anti-STXBantibody, the lower part an immunoblot using anti-Ova antibody. Theintermediates of different steps of the purification procedure areshown. Lane 1: uncoupled proteins (marked by a cross). Lane 2: couplingreaction (coupling product marked by an arrow). Lane 3: eluate of theimmunoaffinity column doted with anti-STxB antibody. Lanes 4-7:fractions from the gelfiltration column. Lane 4: fractions 9-10. Lane 5:fractions 11-12. Lane 6: fractions 13-14. Lane 7: fractions 15-19 (freeSTxB-Cys). Fractions 11-12 contain the bulk of monomeric couplingproduct. Some material with lower electrophoretic mobility can also bedetected, originating likely from dimeric Ova present in the originalpreparation.

[0088]FIG. 8 represents immunofluorescence analysis of STxB-Cys-Ovatransport in HeLa cells. The coupling product STxB-Cys-Ova (upper partof the figure) or a mixture of STxB-Cys and Ova (lower part of thefigure) were incubated with HeLa cells on ice. After washing, the cellswere shifted for 45 min to 37° C., fixed, and stained with the indicatedantibodies. Note that when Ova is linked to STxB-Cys (top), the proteinis vectorized into the Golgi apparatus, co-stained for the Golgi markerRab6. If Ova is only mixed with STxB-Cys (bottom), the protein cannot bedetected on the cells.

[0089]FIG. 9 shows MHC class 1 and 11 restricted antigen presentationinduced by incubation of D1 cells with STxB-Cys-Ova. See text fordetails.

EXAMPLE 1 Preparation of the universal carrier

[0090] a) Construction of a Plasmid Expressing STxB-Cys:

[0091] In a preferred embodiment, the plasmid pSU108 (7) was modified tointroduce the Cysteine codon tgt at the 3′ end of the B-fragment cDNA.PCR primer A: SEQ ID no 3 (5′-AGCGMGTTATTTTTCGTTGTTGACTCAGAATAGCTC-3′)and primer A′: SEQ ID no 4 (5′-GAGCTATTCTGAGTCAACACGAAAATMCTTC-3′) wereused with plasmid specific primers ShigaAtpE: SEQ ID no 5(5′-CACTACTACGTTTTMC-3′) and Shiga-fd: SEQ ID no 6(5′-CGGCGCAACTATCGG-3′) to produce DNA fragments which, in a second PCRwith primers Shiga AtpE and Shiga-fd yielded a fragment that was clonedinto the SphI and SalI restriction sites of pSU108. Sequences derived byPCR were verified by dideoxy-sequencing.

[0092] b) Protein Purification:

[0093] b) 1. Preparation of the Periplasmic Extract was Performed asFollows:

[0094] inoculate 125 ml of LB/Amp with 125 μl of an overnight culturegrown at 30° C.,

[0095] grow over night at 30° C.,

[0096] transfer into 375 ml of LB/Amp at 50° C.; incubate 4 hours at 42°C.,

[0097] centrifuge to pellet cells,

[0098] wash cells 3 times with 10 mM Tris/HCl, pH 8.0,

[0099] re-suspend cells in 200 ml of 25% sucrose, 1 mM EDTA, 10 mMTris/HCl, Ph 8.0; incubate at room temperature for 10 min.,

[0100] centrifuge to pellet cells,

[0101] re-suspend cells in 200 ml of ice cold water containing aprotease inhibitor cocktail; incubate on ice for 10 min.,

[0102] centrifuge; collect supernatant; add 20 mM Tris/HCl, Ph8.0.

[0103] b) 2. Purification on Columns:

[0104] The periplasmic extract was loaded on a QFF anion exchangercolumn (pharmacia) and eluted at 230 mM Nacl. STxB-Cys containingfractions are pooled, diluted 4-fold and loaded on a Mono Q anionexchanger column (pharmacia), followed by elution at 230 mM Nacl. Afterconcentration with microconcentration devices from PallFiltron, thepooled fractions were passed through a Sephadex 75 gel filtrationcolumn. Purity was above 95% (FIG. 1).

[0105] b) 3. Product Characterization:

[0106] The B-fragments of STxB-Cys, purified from Sephadex 75 gelfiltration columns, are essentially monomeric (FIG. 1). This is inmarked difference to constructions where the Cysteine was added at morethan 2 amino acids from the natural C-terminus of the B-fragment. Inthese cases, neighbouring B-fragments within a pentamer are engaged indisulfide bonds.

EXAMPLE 2 Conditions for Coupling of Activated Peptides to the UniversalCarrier

[0107] a) Carriers:

[0108] Three different carriers have been compared.

[0109] 1) STxB-Cys: B-fragment to which a Cys has been added right toits C-terminus. This protein elutes as a monomer from the purificationcolumns.

[0110] 2) STxB-Z₂-Cys: carrier with a short spacer (2 amino acidsresulting from a cloning cassette) between the C-terminus of the wildtype B-fragment and the Cys. The majority of the protein eluted asdimers from the purification columns. These can be separated underreducing conditions, indicating the formation of disulfide bonds betweenmonomers in the pentameric B-subunit complex.

[0111] 3) STxB-Glyc-Cys-KDEL: carrier in which the Cys is locatedbetween a Glycosylation cassette being 9 amino acid long and aC-terminal KDEL peptide. The majority of the protein eluted as dimersfrom the purification columns. These can be separated under reducingconditions, indicating the formation of disulfide bonds between monomersin the pentameric B-subunit complex.

[0112] b) Test Peptides:

[0113] 1) Pep1: a synthetic peptide of 16 amino acids carrying the SL8antigenic peptide derived from chicken ovalbumin.

[0114] 2) Pep2: a synthetic peptide of 24 amino acids as above with, inaddition, a His-gag at its C-terminus.

[0115] 3) SL8: the antigenic peptide from ovalbumin that can directlyexchange with peptides on MHC class I complexes at the plasma membraneof antigen presenting cells.

[0116] c) Coupling Conditions:

[0117] Under reducing conditions (Type1): Fusion proteins were treatedwith DTT overnight, then activated peptide (carrying a bromo acetategroup at its N-terminus) was added in excess. Conditions used for thefirst coupling experiments using fusion proteins will mostly dimerizemonomers (proteins STxB-Z₂-Cys and STxB-Glyc-Cys-KDEL).

[0118] Under non-reducing conditions (Type2): Fusion proteins aredirectly reacted with the activated peptides.

[0119] d) Biochemical and Morphological Controls:

[0120] Pep2 carries a His-tag. This has allowed us, using an anti-Hisantibody, to show the presence of Pep2 on B-subunit by Western blotting,and the B-subunit dependent transport of Pep2 in HeLa cells.

[0121]FIG. 2a shows that a dose dependent stimulation of the B3Z CTLhybridoma (measurement of β-galactosidase activity) was observed withnon-fixed cells, while fixation abolished antigen presentation.

[0122] Note that antigen presentation only works on non-fixed cells,indicating that the observed presentation does not result fromcontaminating free Pep2.

[0123]FIG. 2b shows the control experiment of FIG. 2a in which it isshown that fixed D1 cells can still present free SL8 peptide.

[0124] In FIG. 3, it appears that in this type of protocol, some freePep1 appears to co-purify with the fusion protein, since at high doses(200-1000 nM), some presentation was observed on fixed cells.Presentation by SL8 is shown to the right.

[0125] In FIG. 4, the coupled protein (lanes 1 and 2) or the SL8 Peptide(lanes 5 and 6) were incubated (lanes 2 and 5) or not (lanes 1 and 4)with anti-B-subunit Fab-fragment derived from the 13C4 antibody whichinhibits the binding of the B-subunit to Gb3. Note that the Fab-fragmentneutralizes the capacity of the B-subunit to introduce the antigenicpeptide into the class I pathway, while the presentation with SL8 is notaffected under these conditions. The background signal in thisexperiment was at 0.3.

[0126] In FIG. 5a, D₁ cells were pre-treated with PPMP (see FIG. 3b of(2)) for 3 days. This treatment lead to an important decrease of Gb3expression at the cell surface, without however eliminating itcompletely. Under this condition, antigen presentation from a couplingreaction of Pep1 with STxB-Glyc-Cys-KDEL was significantly reduced,indicating that Gb3 is important for the presentation phenomena.

[0127] It appears from all these experiments that the coupling undernon-reducing to STxB-Cys is surprisingly efficient (in terms ofsensitivity; note that, as shown in FIG. 1, only 4 nM of STxB-Cys-Pep2are necessary to have a response). Thus, the universal carrier STxB-Cysis preferred due to its simplicity in its preparation and to thereproducibility of the coupling.

[0128] Hence, the optimal conditions for coupling of activated peptidesto STxB-Cys were the following:

[0129] dialyse STxB-Cys against 20 mM Borate buffer, pH 9.0, 150 mMNaCl,

[0130] concentrate to 1 mg/ml,

[0131] dissolve N-terminally activated peptide (activated withbromoacetate anhydride) at 12 mM in DMSO,

[0132] dilute peptide to 0.2 mM in protein solution,

[0133] incubate 12 hours at room temperature,

[0134] dialyse against PBS.

EXAMPLE 3 Characterization of STxB as to its Antigen PresentationCapacity

[0135] The following experimental series will help to fully describe thecapacity of STxB to function in antigen presentation system.

[0136] a) Class I— and Class II-Restricted Antigen Presentation:

[0137] A peptide carrying class I- and II-restricted antigenic peptidesfrom chicken ovalbumin(Br—CH₂—CO—NH-LEQLESIINFEKLTEWSLKISQAVHAAHAEINEAGR, sequences 257-264and 323-339 were coupled to STxB-Cys, and the class I— and classII-restricted presentation of these peptides were assayed using thecorresponding T-cell hybridomas.

[0138] b) Coupling of Whole Size Proteins.

[0139] Our preliminary evidence suggests that chicken ovalbumin can becoupled to STxB-Cys. These experiments have be n done with the SPDPheterobifunctional cross-linker. (Carlsson t al., 1978).

[0140] A first series of antigen presentation experiments indicated thatthe ovalbumin protein can be introduced into the endogenous MHC classI-restricted antigen presentation pathway of mouse dendritic cells. SPDPhas the inconvenience of being cleavable by serum thiolases. Thiscross-linker was successfully substituted by MBS which is non-cleavable.Other antigenic proteins (Mart 1 and polypeptides derived from HPV16-E7and Muc1) are tested to show that the procedure is of universal use.

[0141] c) Coupling of Complex Protein Mixtures.

[0142] A lysate from the cervix carcinoma-derived cell line Caski isused. This cervix carcinoma cell line, which expresses the HLA-A2 alleleat its membrane, also expresses Human papillomavirus derived peptides.E7 is a early transcribed ORF from HPV which is necessary fortransformation of primary keratinocytes. Since anti-E7 HLA A2-restrictedCTL are elicited in vitro. The efficacy of the coupling of this proteinmixture by a presentation assay specific for HLA-A2 E7 derived peptideswas tested. As control, a lysate from a HLA-A2-positive cell line whichdoes not express E7 (croft cells or Daudi) was coupled to STxB-lys.

EXAMPLE 4 Application to MHC class I-Restricted Antigen Presentation

[0143] The experiment of FIG. 4 shows that STxB-Cys dependent antigenpresentation is inhibited when the interaction with Gb₃ is abolished.Here it is found that prebinding of the Fab fragment of monoclonal Abagainst STxB to 0.1 μM STxB-Cys, coupled to SL8, inhibited antigenpresentation, suggesting that STxB-Cys binding to Gb₃ is necessary forantigen presentation. Similar results were obtained when Gb₃-expressionwas inhibited with a drug (FIG. 4).

EXAMPLE 5 Reaction Chain for Coupling Ovalbumine to the STxB-Cys

[0144] The reaction scheme is shown in FIG. 6.

[0145] In a first reaction, the N-hydroxysuccinimide ester moiety of MBSreacts with primary amines on an antigenic target protein, such as themodel protein ovalbumin (Ova). The reaction product is purified and thenincubated in a second reaction with STxB-Cys leading to coupling via themaleimidobenzoyl moiety.

[0146]FIG. 7 shows the SDS-PAGE and Western analysis of a typicalcoupling reaction involving STxB-Cys and Ova. For coupling, 20 mg/ml ofOva in 100 mM HEPES, pH 7.4, was incubated with 4.5 mM of MBS for 30 minat room temperature. The reaction is passed through a PBS/EDTA 10 mMequilibrated gel filtration column. Eluted Ova is concentrated to 20mg/ml. 1 volume of STxB-Cys at 3.5 mg/ml in PBS/EDTA is mixed with 1volume of activated Ova and incubated over night at room temperature.

[0147]FIG. 7 shows that within the coupling reaction, bands with lowerelectrophoretic mobility (labeled with arrows; coupling product) can bedetected in addition to uncoupled STxB (upper part) and uncoupled Ova(lower part; uncoupled proteins are labeled with a cross). The reactionproduct is purified by passage through an immunoaffinity column madewith 13C4 anti-STxB monoclonal antibody (lane IP column). Note that freeOva is eliminated. Eluted STxB-Cys (coupled and non-coupled) is thenpassed through a gel filtration column to separate free STXB (fractions15-19) from coupled STxB-Cys (fractions 9-14; note that fractions 11-12contain the bulk of coupled protein; the upper coupling band, which isminor compared to the lower band, probably results from dimeric Ova).

EXAMPLE 6 Intracellular Transport Characteristics of STxB-Cvs-OvaCoupling Product

[0148] 0.5 μM of STxB-Cys-Ova was incubated with HeLa cells on ice. Thecells were washed and shifted to 37° C. for 45 min, fixed, and stainedfor the indicated antibodies. As shown in FIG. 8, when STxB-Cys and Ovawere linked by MBS, Ova immunoreactivity could be detected together withSTxB immunoreactivity in the Golgi apparatus, stained by Rab6. When bothproteins are incubated as separate ntities with HeLa cells, onlySTxB-Cys is transported to the Golgi, while Ova cannot be detected onthe cells. These data clearly show that couples STxB-Cys is stilltransported in the same manner as uncoupled STxB-Cys, and that Ova isvectorized via STxB-Cys.

EXAMPLE 7 The STxB-Cys Allows Both, MHC Class I and II RestrictedPresentation of Peptides Derived from Full Size Exogenous AntigenicProteins

[0149] In a first experiment (FIG. 9, left), we have shown that whenSTxB-Cys-Ova is used to sensitize the murine dendritic cell line D1, aclear increase of the presentation of the MHC class II restricted Ovaderived peptide Ova₃₂₃₋₃₃₉ is observed, compared to D1 cells pulsed withnon-vectorized Ova. Indeed, a significant presentation of Ova₃₂₃₋₃₃₉peptide could be detected with as little as 0.01 nM of STxB-Cys-Ova,whereas 10 nM of full size Ova was required for an efficientpresentation of the same peptide. The presentation of the IA^(b)restricted Ova₃₂₃₋₃₃₉ peptide was revealed using the B097.10 hybridomathat produces IL-2 after recognition of this peptide. As a control, wedid not observe IL-2 secretion when an irrelevant MHC class IIrestricted hybridoma was used instead of B097.10 (data not shown).

[0150] In a second experiment, we have pulsed the same D1 dendriticH2^(b) restricted cell line with either Ova alone or with STxB-Cys-Ova.No presentation of the Ova-derived immunodominant SL8 peptide(Ova₂₅₇₋₂₆₄) was observed when the D1 cells were sensitized with up to100 nM of free Ova, while 1-10 nM of STxB-Cys-Ova allowed thepresentation of the SL8 peptide, as revealed by the specific B3Zhybridoma that recongnize the SL8 peptide in the context of K^(b)molecules. As a control, it was shown that no activation of anirrelevant hybridoma was observed under the same experimentalconditions.

[0151] Altogether, these results clearly demonstrate that STxB-Cystargets full size proteins with high efficiency into both, the MHC classI and class II pathways.

BIBLIOGRAPHY

[0152] (1) Ren-Shiang Lee, Eric Tartour, Pierre van der Bruggen, ValerieVantomme, Isabelle Joyeux, Bruno Goud, Wolf Herman Fridman and LudgerJohannes, “Major histocompatibility complex class I Presentation ofexogenous soluble tumor antigen fused to the B-fragment of Shiga toxin”.Eur. J. Immunol. (1998) 28: 2726-2737.

[0153] (2) Nacilla Haicheur, Emmanuelle Bismuth, Sophie Bosset, OlivierAdotevi, Guy Warnier, Valerie Lacabanne, Armelle Regnault, CatherineDesaymard, Sebastian Amigorena, Paola Ricciardi-Castagnoli, Bruno Goud,Wolf H. Fridman, Ludger Johannes and Eric Tartour, “The B Subunit ofShiga Toxin Fused to a Tumor Antigen Elicits CTL and Targets DendriticCells to Allow MHC Class I-Restricted Presentation of Peptides Derivedfrom Exogenous Antigens”. The Journal of Immunology (2000) 165:3301-3308.

[0154] (3) Noakes, K. L., H. T. Teisseranc, J. M. Lord, P. R. Dunbar, V.Cerundolo and L. M. Roberts “Exploiting retrograde transport ofShiga-like toxin 1 for the delivery of exogenous antigens into the MHCclass I presentation pathway”. Febs. Lett. (1999) 453:95.

[0155] (4) Philippe Schelté, Christophe Boeckler, Benoit Frisch andFrancis Schuber “Differential Reactivity of Maleimide and Bromoacetylfunctions with Thiols: Application to the Preparation of LiposomalDiepitope Constructs”. Eur. J. Immunol. (1999) 29:2297-2308.

[0156] (5) Carlsson, J., H. Drevin, and R. Axen. 1978. Proteinthiolation and reversible protein-protein conjugation. N-Succinimidyl3-(2-pyridyldithio)propionate, a new heterobifunctional reagent.Biochem. J. 173:723-737.

[0157] (6) Su, G. F., H. N. Brahmbhatt, J. Wehland, M. Rohde and K. N.Timmis “Construction of stable LamB-Shiga toxin B subunit hybrids:analysis of expression in Salmonella typhimurium aroA strains andstimulation of B subunit-specific mucosal and serum antibody responses”.(1992) Infect. Immun. 60:3345-3359.

[0158] (7) Johannes, L., Tenza, D., Antony, C. and Goud, B., “Retrogradetransport of KDEL-bearing B-fragment of Shiga toxin”. (1997) J. Biol.Chem. 272: 19554-19561.

[0159] (8) N. A. Stockbine, M. P. Jackson, L. M. Sung, R. K. Holmes, A.D. O'Brien, J Bacteriol 170, 1116-22 (1988).

1 7 1 90 PRT Artificial Sequence Description of Artificial SequenceUniversal carrier 1 Met Lys Lys Thr Leu Leu Ile Ala Ala Ser Leu Ser PhePhe Ser Ala 1 5 10 15 Ser Ala Leu Ala Thr Pro Asp Cys Val Thr Gly LysVal Glu Tyr Thr 20 25 30 Lys Tyr Asn Asp Asp Asp Thr Phe Thr Val Lys ValGly Asp Lys Glu 35 40 45 Leu Phe Thr Asn Arg Trp Asn Leu Gln Ser Leu LeuLeu Ser Ala Gln 50 55 60 Ile Thr Gly Met Thr Val Thr Ile Lys Thr Asn AlaCys His Asn Gly 65 70 75 80 Gly Gly Phe Ser Glu Val Ile Phe Arg Cys 8590 2 270 DNA Artificial Sequence Description of Artificial SequencePolynucleotide 2 atgaaaaaaa cattattaat agctgcatcg ctttcatttt tttcagcaagtgcgctggcg 60 acgcctgatt gtgtaactgg aaaggtggag tatacaaaat ataatgatgacgataccttt 120 acagttaaag tgggtgataa agaattattt accaacagat ggaatcttcagtctcttctt 180 ctcagtgcgc aaattacggg gatgactgta accattaaaa ctaatgcctgtcataatgga 240 gggggattca gcgaagttat ttttcgttgt 270 3 37 DNA ArtificialSequence Description of Artificial Sequence Primer A 3 agcgaagttatttttcgttg ttgactcaga atagctc 37 4 33 DNA Artificial SequenceDescription of Artificial Sequence Primer B 4 gagctattct gagtcaacacgaaaaataac ttc 33 5 17 DNA Artificial Sequence Description of ArtificialSequence Primer A′ 5 cactactacg ttttaac 17 6 15 DNA Artificial SequenceDescription of Artificial Sequence Primer B′ 6 cggcgcaact atcgg 15 7 36PRT Artificial Sequence synthetic peptide derived from chicken ovalbumin7 Leu Glu Gln Leu Glu Ser Ile Ile Asn Phe Glu Lys Leu Thr Glu Trp 1 5 1015 Ser Leu Lys Ile Ser Gln Ala Val His Ala Ala His Ala Glu Ile Asn 20 2530 Glu Ala Gly Arg 35

1. Universal polypeptidic carrier for targeting directly or indirectly amolecule to Gb3 receptor expressing cells and having the followingformula STxB-Z(n)-Cys, wherein: STxB is the Shiga Toxin B subunit or afunctional equivalent thereof, Z is an amino-acid devoided of sulfydrylgroup, n being 0, 1 or a polypeptide, Cys is the amino-acid Cysteine. 2.Universal carrier according to claim 1 wherein n is
 0. 3. Universalcarrier according to claim 1 or 2 wherein the molecule is selected inthe group constituted of proteins, peptides, oligopeptides,glycoproteins, glycopeptides, nucleic acids, polynucleotides, or acombination thereof.
 4. Universal carrier according to claim 1 or 2wherein the molecule is covalently linked to the —S residue of theuniversal carrier by a —S—S—, or —S—CO—, or —S—CH₂—. Or —S—NH— linkage.5. Universal carrier according to claim 4 wherein the molecule is anantigen to be targeted to antigen presentating cells.
 6. Universalcarrier according to claim 1 or 2 wherein the universal carrier iscovalently linked to an oligopeptide or a polypeptide by a —S—S—, or—S—CO—, or —S—CH₂— or —S—NH— linkage, and the molecule to be targeted isoperably linked to the said oligopeptide or polypeptide.
 7. Universalcarrier according to claim 6 characterized in that it is covalentlylinked to a poly-lysine oligopeptide and the molecule to be targeted isa nucleic acid or an oligonucleotide operably linked to the saidpoly-lysine moiety.
 8. Universal carrier according to claim 4 whereinthe molecule is a cytotoxic drug or pro-drug to be targeted to tumorcells expressing Gb3 receptor.
 9. An isolated polynucleotide selectedfrom the group consisting of: a) a polynucleotide comprising thenucleotide sequence STxB encoding the Shiga Toxin B subunit or afunctional equivalent thereof bearing at its 3′end the codon TGT, or thecodon TGC encoding Cysteine, b) a polynucleotide comprising a nucleotidesequence having at least 80% sequence identity to a nucleotide sequenceencoding the Shiga Toxin B subunit or a functional equivalent thereofbearing at its 3′end the codon TGT or TGC, c) a nucleotide sequencecomplementary to the sequence in a) or b).
 10. A polynucleotideaccording to claim 9 having the SEQ ID NO
 2. 11. A recombinant vector,or plasmid, comprising a polynucleotide sequence according to claim 9 or10, for the expression of the universal vector of claim 1 in anappropriate host cell.
 12. A recombinant cell line obtained bytransformation with the recombinant vector according to claim
 11. 13. Arecombinant cell line according to claim 12 being a procarytic cellline, preferably E. coli.
 14. A recombinant cell line according to claim11, 12 or 13, deposited at CNCM on Dec. 19, 2000, with the registrationnumber I-2604.
 15. A method for constructing a recombinant vectoraccording to claim 11 or 12 comprising: a) providing a plasmidcomprising STxB sequence; b) applying two PCR amplification steps usingtwo couples of primers, AA′ and BB′, A and B being complementary to eachother and comprising the Cys codon, A′ and B′ being outside the STxBsequence; c) isolating the amplified fragments; d) hybridizing theamplified fragments; e) applying a PCR amplification on the hybridizedfragments; f) insertion of the amplified fragment into a plasmid.
 16. Amethod according to claim 15 wherein in step f) the fragment is insertedinto the SphI and SalI restriction site of the plasmid pSU108.
 17. Aprocess for producing a purified polypeptide according to claim 1comprising: a) culturing the cell line according to anyone of claim 12to 14, b) obtaining a periplasmic extract of said cells c) purifyingsaid polypeptide.
 18. A process according to claim 17 wherein in a), thecell line is E. coli and in c) the purification is made by anionexchange column chromatography followed by a gel filtration columnchromatography.
 19. Method for delivering an sequence of interest intothe MHC class I and MHC class II pathway using the a product obtained bycovalent binding of the Cys moiety of the universal carrier with saidsequence of interest.
 20. Method for delivering an expression vectorcontaining a sequence of interest into a Gb3 receptor expressing cellscharacterized in that said expression vector is operably linked to alysinerich peptide covalently linked to the Cys moiety of the universalcarrier.
 21. Method according to claim 20 wherein the lysine richpeptide is a 16-mer poly-lysine.
 22. Method according to claim 19 to 21wherein the sequence of interest is selected amongst: a sequenceencoding an immunogenic peptide, or a sequence encoding a drug or apro-drug becoming toxic for the Gb3 receptor expressing cells, or asequence encoding a therapeutic active molecule.
 23. Pharmaceuticalcomposition for enhancing the immunogenicity of a peptide or a proteinor a glycoprotein or a lipopeptide, containing the polypepidic carrieraccording to claim 1 or 2 covalently linked by its Cys moiety to saidpeptide or protein or glycoprotein or lipopeptide.
 24. Pharmaceuticalcomposition for treating tumor cells bearing the Gb3, containing thepolypeptidic carrier according to claim 1 or 2 covalently linked by itsCys moiety to a drug or a prodrug toxic for said tumor cells.